The p53 tumor suppressor protein can induce both apoptosis andcell
cycle arrest. Moreover, we and others have shown previouslythat p53 is
a potent mediator of differentiation. For example,expression of
ptsp53, a temperature-inducible form of p53, inducesdifferentiation of
leukemic monoblastic U-937 cells. The functionsof p53 have for long
been believed to be dependent on the transactivatingcapacity of p53.
However, recent data show that both p53-inducedcell cycle arrest and
apoptosis can be induced independentlyof p53-mediated transcriptional
activation, indicating alternativepathways for p53-induced apoptosis
and cell cycle arrest. Thebcl-2 proto-oncogene
contributes to the development of certainmalignancies, probably by
inhibition of apoptosis. Interestingly,Bcl-2 has been shown to inhibit
p53-mediated apoptosis as wellas p53-mediated transcriptional
activation. Asking whether Bcl-2would interfere with the p53-mediated
differentiation of U-937cells, we stably transfected
bcl-2 to U-937 cells induciblyexpressing p53.
Although the established Bcl-2-expressing cloneswere resistant to
p53-mediated apoptosis, we did not observeany interference of Bcl-2
with the p53-mediated differentiation,suggesting separable pathways
for p53 in mediating apoptosisand differentiation of U-937 cells.
Neither did expression ofBcl-2 interfere with p53-induced expression
of endogenous p21,suggesting that p53-induced differentiation might be
dependenton the transcriptional activity of p53. To further
investigatewhether the p53-mediated differentiation of U-937 cells
dependson the transcriptional activity of p53, we overexpressed
trans-activation-deficientp53, a transcriptionally
inactive p53 mutant in these cells.However, in contrast to the effects
of wild-type p53, expressionof trans-activation-deficient
p53 did neither induce signs ofapoptosis nor of differentiation in
U-937 cells. Our resultsindicate that the transcriptional activity of
p53 is essentialboth for p53-mediated apoptosis and differentiation of
U-937cells.

Introduction

One of the key proteins protecting tissues from malignant
transformationis the tumor suppressor p53 (1)
. The
tumor-suppressing activityof p53 is usually explained by its ability
to prevent expansionof potentially malignant cells by either induction
of apoptosisor an arrest in the G1 phase of the
cell cycle (1, 2, 3)
. p53is a transcription factor and can
transactivate genes of importanceboth for apoptosis (i.e.,
bax; Ref. 4
) and G1 arrest
(i.e.,p21; Refs. 2
and 5
).

Apart from its role in apoptosis and cell cycle regulation,p53 has
also been shown to participate in the differentiationprocess of a
number of tissues such as pancreatic carcinomacells, muscle cells,
keratinocytes, neurons, thyroid cells (6,7, 8)
, and various
hematopoietic cell lines. For example, expressionof p53 induces
differentiation of leukemic L12 pre-B-cells,erythroleukemic K562
cells, Friendvirus transformed erythroleukemiccells, and promyelocytic
HL-60 cells (9, 10, 11, 12)
. Our ownresults show that inducible
overexpression of p53 induces differentiationper se as well
as promotes induction of differentiation withVit
D33
in monoblastic U-937 cells (13)
. The molecular mechanisms
forp53-mediated differentiation are not clear but do not seem torely
on induction of p21 (14)
or on a cell cycle arrest
mediatedby the hypophosphorylated form of the retinoblastoma protein
(15)
.

The proto-oncogene bcl-2 was first identified in the
t(14;18)chromosomal translocation involved in 70% of human follicular
B-celllymphomas (16, 17, 18)
. Overexpression of Bcl-2 in
lymphoidcells in culture and in transgenic mice prevents apoptosis
inducedwith a wide variety of agents, such as steroids, gamma
irradiation,or growth factor deprivation (19, 20)
.
Importantly, Bcl-2has also been shown to inhibit p53-induced apoptosis
(21, 22,23)
. Besides its antiapoptotic properties, Bcl-2 has
a cellcycle-inhibitory function separable from its promotion of cell
survival(24, 25, 26)
. Although levels of Bcl-2 fall during
myeloiddifferentiation of normal bone marrow cells, consistent withan
inhibitory role for Bcl-2 in hematopoietic differentiation
(27)
,Bcl-2 does not seem to be a potent inhibitor of
induced myeloiddifferentiation. Hence, overexpression of Bcl-2 in
HL-60 cellsdoes not inhibit differentiation induced with
all-trans retinoicacid (19, 28)
, and
neutrophils from transgenic mice overexpressingBcl-2 do not have a
defect differentiation (29)
.

Several mechanisms for Bcl-2-mediated inhibition of p53 activityhave
been reported. For example, Bcl-2 binds to the proteinproduct from the
p53-target gene bax and inhibits its death-inducingactivity
(4, 23, 30)
. Moreover, Bcl-2 inhibits nuclear importof
p53 in some cells (31)
. High levels of Bcl-2 can also
inhibitthe induction of the p53-regulated genes p21,
bax, and gadd45after genotoxic stress
(32)
. However, Bcl-2 has been shownnot to interfere with
p53-mediated G1 arrest (22)
.
Interestingly,the relationship between p53 and Bcl-2 seems reciprocal
in thatp53 can transcriptionally repress the expression of Bcl-2
(4, 23, 33, 34)
.

Both p53-mediated cell cycle arrest (35, 36)
and apoptosis
(37,38, 39)
can be induced independently of the
transcriptionalactivity of p53. For example, by means of a potential
SH3-domainbinding site, p53 has been suggested to participate in a
growth-arrestingsignal transduction pathway (35, 36)
.

Given the ability of Bcl-2 to inhibit p53-mediated apoptosis,
potentiallyby means of its reported inhibitory effect on p53-mediated
transactivation(32, 40)
, we set out to determine whether
expression of Bcl-2would interfere with p53-mediated
differentiation. We show thatBcl-2 does not interfere with
p53-mediated differentiation orwith the transcriptional activity of
p53. To further investigatewhether the transactivating properties of
p53 are necessaryfor p53-mediated differentiation, we expressed a
temperature-sensitivetranscriptionally inactive mutant of p53
[i.e., p53(25, 26,Val135)] (Ref. 41
) in U-937
cells. Our results show that thetranscriptional activity of p53 is
essential for induction ofdifferentiation of U-937 cells.

Results

The Cell Death and Proliferation-related Properties of
ptsp53(Val135) Depend on Small Shifts of the Temperature.
Neither p53 protein (42, 43, 44)
nor mRNA
(44)
can be detectedin monoblastic U-937 cells, whose
gene for p53 is altered bya point mutation (45)
.
Therefore, to obtain inducible expressionof p53 on a p53-null
background, we overexpressed a temperature-inducibleform of p53
[(i.e., ptsp53(Val135)] in U-937 cells as described
previously(13)
. When incubated at 32°C, U-937/ptsp53/A2
cells showsigns of differentiation, cell cycle arrest, and apoptosis
(13)
,reflecting the wild-type p53 activity of ptsp53.
However, adifference in ptsp53 function was observed during induction
ofwild-type p53 activity of ptsp53 by incubation of U-937/ptsp53/A2
cellsat temperatures around 32°C. When incubated at 32.5°C,mainly
p53-mediated differentiation (data not shown) with almostno signs of
cell death was observed, whereas incubation at 31.5°Cinduced
pronounced p53mediated cell death measured both by trypanblue
exclusion (Fig. 1)
and as fraction of cells with a sub-G1DNA
content (Table 1)
. Because this difference in p53 functionmight suggest that ptsp53
achieves a higher wild-type p53 activitywhen incubated at 31.5°C
than at 32.5°C, we wanted todetermine whether this functional
difference extended to thecell cycle regulatory properties of p53. For
this purpose, mock-transfectedand ptsp53-expressing U-937 cells were
incubated at 37°C,32.5°C, and 31.5°C. Each day, cells were
harvested andsubjected to a cell cycle analysis by flow cytometry
(Table2). Interestingly, after 24 h, the fraction of
ptsp53-expressingcells present in S-phase after incubation at 31.5°C
waslower than the S-phase fraction of ptsp53-expressing cells
incubatedat 32.5°C in repeated experiments. However, this
temperature-relateddifference disappeared with prolonged culture,
because the cellcycle distributions of U-937/ptsp53/A2 cells incubated
at 31.5°Cand 32.5°C for 48 and 72 h, respectively,
were comparable.To investigate whether the initial cell cycle
regulatory differencesof ptsp53 were reflected by an altered
p53-mediated up-regulationof the cell cycle regulator p21, a Western
blot was performedin parallel with the cell cycle analysis made after
24 h. Nop21 was detected in a mock-transfected control clone or
in theU-937/ptsp53/A2 clone incubated at 37°C (i.e., the
nonpermissivetemperature; Fig. 2
). However, when incubated at 32.5°Cand 31.5°C, p21 was
up-regulated at comparable levels inthe U-937/ptsp53/A2 clone.
Moreover, a slightly higher levelof p21 protein in the 31.5°C
incubation was observed onrepeated Western blots, further supporting
that ptsp53 has ahigher wild-type p53 activity at 31.5°C than at
32.5°C.To investigate whether this elevated level of p21 protein
reflecteda higher transactivating capacity of p53 at the p21 promoter,
luciferasereporter experiments were performed. Mock-transfected and
ptsp53expressingU-937 cells were transfected with the firefly
luciferase geneunder the control of the p21 promoter. The firefly
luciferasegene under the control of the SV40 promoter was serving as a
positivecontrol of luciferase activity. After incubation at 32.5°C
and31.5°C for 16 h, the luciferase activity of the cells was
determined(Table 3)
. As measured by luciferase activity, the p53-mediatedtransactivation
of the p21 promoter was slightly higher at 31.5°Cas compared with
32.5°C, possibly indicating a higher wild-typep53 activity at
31.5°C. Therefore, to obtain maximal wild-typep53 activity, all
viability studies and transactivation studiesin subsequent experiments
were performed at 31.5°C. However,because pronounced cell death
makes it difficult to performdifferentiation experiments, all
differentiation studies wereperformed at 32.5°C.

Table 1 Percentage of ptsp53-expressing U-937 cells present in the
sub-G1 phase of the cell cycle after incubation at
different temperatures

Control and U-937/ptsp53 A2 cells were seeded at 200,000 cells/ml in
culture medium and incubated at 37°C, 32.5°C, and 31.5°C. After
48 h, viability was assessed by trypan blue exclusion.
Concomitantly, cells were subjected to cell cycle analysis by flow
cytometry as described in "Materials and Methods." Mean values from
three experiments are shown. Viability of control cells was always
>95%, and the fraction of control cells with a sub-G1 DNA
was always <10%.

Cells were seeded in culture medium at 200,000 cells/ml and incubated
at the indicated temperatures. On days 1, 2, and 3, cells were explored
for cell cycle distribution by a FACS analysis. Values show percentage
of viable cells (i.e., cells with sub-G1 DNA
content are excluded). One representative experiment is shown.

Fig. 2. Temperature dependence of the levels of ptsp53-induced p21. The
U-937/ptsp53 clone A2 and the mock-transfected U-937 clone M2 were
incubated at 31.5°C, 32.5°C (i.e., temperatures
permissive for wild-type p53 activity), and 37°C
(i.e., the nonpermissive temperature for wild-type p53
activity). After 24 h, cells were subjected to Western blot using
the mouse moAb anti-p21 WAF-1 Ab-1 and an actin antibody, serving as a
control for equal loading (described in "Materials and Methods").
Arrows on the right, positions of the p21
and actin proteins. Left, positions of molecular weight
standards (in thousands). *, the relative amount of p21
protein was estimated by densitometry as described in "Materials and
Methods" and normalized to the amount of actin in the corresponding
lane.

Table 3 Temperature dependence of the ptsp53-induced transcriptional activation
of the p21 promoter in U-937 cells

Mock-transfected and ptsp53-expressing U-937 cells were transfected
with luciferase expression constructs driven by the p21 promoter. The
SV40 promoter was serving as a control of luciferase activity. Cells
were incubated at 32.5°C and 31.5°C, respectively, and after
16 h, the luciferase activity was determined as described in
"Materials and Methods." Values represent mean values from three
independent experiments, each consisting of a triplicate of samples.

p53-mediated Cell Death Is Partially Overrun by High Levels of
Bcl-2.
To study the role of Bcl-2 in p53-mediated differentiation,stable
Bcl-2 overexpression was established in the U-937/ptsp53/A2clone
(13)
. Transfection of the U-937/ptsp53/A2 clone with
bcl-2resulted in 10 clones growing under selective
conditions. Whenanalyzed for expression of Bcl-2 protein by
IP-Western, allof these clones showed a clear overexpression of Bcl-2
as comparedwith wild-type U-937 cells (Fig. 3). To ascertain that clonesstill expressed high levels of
p53, they were analyzed for expressionof p53 by biosynthetic labeling,
IP, and fluorography (datanot shown). On the basis of their high
expression levels ofboth p53 and Bcl-2, clones A4, A8, and A9 were
chosen for furtherexamination.

Fig. 3. Expression of Bcl-2 protein in transfected U-937/ptsp53 cells.
Bcl-2-transfected U-937/ptsp53 clones and wild-type U-937 cells were
subjected to IP, followed by Western Blot (IP-Western) using the mouse
monoclonal anti-bcl-2-antibody 1550 as described in "Materials and
Methods." Arrow on the right, position
of Bcl-2 protein at Mr 26,000. Low amounts
of Bcl-2 protein were detected in U-937 wild-type cells.
Left, positions of molecular weight standards (in
thousands).

To examine whether Bcl-2 can rescue U-937 cells from p53-mediatedcell
death, U-937/ptsp53/Bcl-2 clones and mock-transfected U-937/ptsp53
cloneswere incubated at the optimal temperature for the
apoptosis-inducingactivity of p53 (i.e., 31.5°C) for 4
days. Each day, cellswere counted, and the viability was assessed by
trypan blueexclusion. As shown in Fig. 4
, Bcl-2 conferred partial resistanceto p53-mediated cell death. On day
2, most Bcl-2-expressingcells were alive, in contrast to control cells
already showingpronounced p53-mediated cell death. On day 4, the
viabilityof U937/ptsp53/bcl-2 cells was 45%, still obviously
higher thancontrol cells. To determine whether the cell death was
attributableto apoptosis, the cells were analyzed for expression of
AnnexinV by FACS analysis, concomitantly with propidium iodide
staining(Table 4)
, providing a selective method for detection of apoptosis
(46)
.As demonstrated, the cells showed characteristics of
apoptosis,as measured by expression of Annexin V.

U-937 cells were incubated at an initial concentration of 200,000
cells/ml in culture medium at 31.5°C. After 1 day, cells were
subjected to analysis of Annexin V by flow cytometry. Values shown are
the percentages of cells expressing Annexin V. One representative
experiment of three is shown.

Overexpression of Bcl-2 Does Not Affect the Proliferation Rate of
U-937 Cells.
Because Bcl-2 has been shown previously to possess growth-arresting
features(24, 25, 26)
, we asked whether U-937 cells
transfected withBcl-2 showed a decreased proliferation rate.
U-937/ptsp53/Bcl-2cells and mock-transfected U-937/ptsp53 cells were
incubatedat the temperature nonpermissive for wild-type p53 activity
(i.e.,37°C) for 4 days. Each day, cells were counted, and
viabilitywas assessed by trypan blue exclusion. No difference in
proliferationrate was seen between Bcl-2-expressing and
mock-transfectedU-937/ptsp53 clones, indicating that Bcl-2 does not
influencethe proliferation rate of U-937 cells (Fig. 5)
.

Fig. 5. Growth rate in suspension culture of Bcl-2-expressing cells.
Mock-transfected and Bcl-2-expressing U-937/ptsp53 cells at an initial
concentration of 0.2 x 106 cells/ml were grown in
suspension culture at 37°C (i.e., the temperature
nonpermissive for wild-type p53 activity) for 4 days. The total number
of cells and viability, as judged by trypan blue exclusion, was
determined daily. , Bcl-2/A4; , Bcl-2/A8; , Bcl-2/A9; ,
Mock 2; , Mock 5; , Mock 6. Mean values are from at least three
separate experiments. Viability was always >90% throughout the
experiments.

Preserved p53-mediated Differentiation in the Presence of Bcl-2.
U-937 cells are known to respond with signs of differentiation,when
incubated with substances like Vit D3 (47)
. To investigate
whetherBcl-2 interferes with the Vit D3-induced differentiation of
U-937cells, mock-transfected and Bcl-2-expressing U-937/ptsp53 clones
wereincubated with and without Vit D3 at the temperature nonpermissive
forwild-type p53 activity (i.e., 37°C). NBT reduction
testprovides a functional assessment of myeloid differentiation,
reflectingthe capacity of the cells for respiratory burst. No
differencein NBT reduction was observed between mock-transfected and
Bcl-2-expressingU-937/ptsp53 clones, indicating that Bcl-2 does not
interferewith the induced differentiation of U-937 cells in the
absenceof wild-type p53 activity (Fig. 6). However, Bcl-2 can inhibitthe apoptosis-inducing aspects
of p53. Therefore, we asked whetherBcl-2 would inhibit p53-mediated
differentiation as well. Forthis purpose, Bcl-2-expressing and
mock-transfected U-937/ptsp53clones, as well as mock-transfected
wild-type U-937 cells, wereincubated at the optimal temperature for
the differentiation-inducingactivity of ptsp53 (i.e.,
32.5°C). As measured by reductionof NBT, mock-transfected U-937
cells not expressing ptsp53 showedalmost no signs of differentiation
(Fig. 7). However, U-937clones coexpressing ptsp53 and
Bcl-2 responded with reductionof NBT at levels comparable with control
clones expressing ptsp53alone. This suggests that Bcl-2 does not
interfere with differentiationinduced by p53 per se.
Furthermore, in experiments with differentconcentrations of Vit D3,
the Bcl-2-expressing U-937/ptsp53clones responded with reduction of
NBT at levels comparablewith the ptsp53-expressing control clones
(Fig. 8)
, again supportingthe conclusion that Bcl-2 does not interfere with
the p53-mediateddifferentiation of U-937 cells.

Fig. 7. Effects of Bcl-2 on p53-mediated differentiation, assayed by the
capacity to reduce NBT. Mock-transfected wild type (-
Control) and ptsp53-expressing (+ Control) U-937
cells as well as U-937 cells coexpressing ptsp53 and Bcl-2
(Bcl-2) were incubated at 32.5°C (i.e., the
permissive, differentiation-inducing ptsp53 temperature) at an initial
concentration of 0.2 x 106 cells/ml. After 6 days of
incubation, the cells were subjected to an NBT test. Mean values from
at least four separate experiments are shown; bars,
SE.

Fig. 8. Effects of Bcl-2 on the Vit D3-facilitated p53-induced differentiation,
assayed by the capacity to reduce NBT. Mock-transfected
(Control) and Bcl-2-expressing (Bcl-2)
U-937/ptsp53 cells at an initial concentration of 0.2 x
106 cells/ml were incubated with or without Vit D3 at
different concentrations at 32.5°C (i.e., the
temperature permissive for ptsp53-mediated differentiation). After 4
days, the cells were subjected to an NBT test. The percentage of
NBT-reducing cells was determined by counting 200 cells. , culture
medium; , Vit D3 1 nM; , Vit D3 10 nM.
Mean values from at least four separate experiments are shown;
bars, SE.

p53 Transactivates p21 in U-937 Clones Overexpressing Bcl-2.
Bcl-2 can inhibit the transcriptional activity of p53 in somecell
lines (32, 40)
. Therefore, to determine whether Bcl-2
inhibitsthe transcriptional activity of p53 in U-937 cells,
mock-transfectedand Bcl-2-expressing U-937/ptsp53 clones were
incubated at thepermissive (i.e., 31.5°C) and
nonpermissive (i.e., 37°C)temperature. After 24 h,
cells were subjected to analysis ofp53-mediated transactivation of p21
by biosynthetic labeling,IP, and fluorography (Fig. 9)
. As demonstrated, no p21 is expressedin mock-transfected or
Bcl-2-expressing U-937/ptsp53 clonesincubated at the nonpermissive
temperature. However, at thepermissive temperature, both
mock-transfected and Bcl-2-expressingU-937/ptsp53 clones respond with
up-regulation of p21 at comparablelevels. The up-regulation of p21 is
not attributable to theincubation at 31.5°C, because incubation of
wild-type U-937cells at the permissive temperature does not provoke
expressionof p21 (data not shown; Ref. 15
). These data
indicate thatBcl-2 does not interfere with the transcriptional
activity ofp53 in U-937 cells.

Fig. 9. Effects of Bcl-2 on p53-mediated transactivation of p21 in U-937 cells.
Mock-transfected and Bcl-2-expressing U-937/ptsp53 clones at an initial
concentration of 0.2 x 106 cells/ml were grown in
suspension culture at 31.5°C (i.e., the temperature
permissive for wild-type p53 activity) and 37°C (i.e.,
the nonpermissive temperature for wild-type p53 activity). After
24 h, cells were biosynthetically labeled with
[35S]methionine/cysteine and subjected to IP with the
anti-p21 mouse moAb sc-817. The IP was followed by fluorography, as
described in "Materials and Methods." Arrow on the
right, position of p21 protein. Left,
positions of molecular weight standards (in thousands).

Characterization of Cell Clones Expressing a Transcriptionally
Inactive p53-Mutant in U-937 Cells.
To study the role of transcriptional activity of p53 for p53-mediated
differentiation,TADp53, a transcriptionally inactive form of the
murine temperature-sensitiveptsp53(Val135) (Ref. 41
) was
transfected into U-937 cells,resulting in 36 clones growing under
selective conditions. Fourof these clones were shown to express TADp53
by biosyntheticlabeling, IP, and fluorography (Fig. 10)
and also by repeatedWestern blots (data not shown). These four clones
were chosenfor further investigation and were designated U-937/TADp53
C5,C14, C31, and C35.

Fig. 10. Expression of transfected TADp53 protein in U-937 cells. Control and
TADp53-transfected clones were biosynthetically labeled with
[35S]methionine/cysteine and subjected to IP with the
anti-p53 mouse moAb Ab-1 and the anti-p21 mouse moAb WAF-1 Ab-1,
serving as a negative control. The IP was followed by fluorography, as
described in "Materials and Methods." TADp53-expressing clones C5,
C14, C31, and C35, the ptsp53-expressing clone A2, and the
mock-transfected clone M1 are shown. Arrow on the
right, position of p53 protein. Left,
positions of molecular weight standards (in thousands).

Because recent reports have shown that TADp53 causes a slight
activationof reporter constructs carrying a p53 promoter sequence
(48, 49), we analyzed the protein levels of the endogenous
p53 targetgene p21 in response to expression of wild-type
TADp53 by Westernblot. As shown in Fig. 11
, no p21 is expressed in U-937/ptsp53/A2cells at the nonpermissive
temperature (i.e., 37°C), butwhen incubated at the
permissive temperature (i.e., 31.5°C),the cells respond
with a pronounced up-regulation of p21. However,U-937 cells expressing
TADp53 show no detectable up-regulationof p21 when incubated at the
temperature permissive for wild-typep53-activity. Thus, TADp53 does
not induce detectable expressionof endogenous p21 protein in U-937
cells as estimated by Westernblot, confirming its defective
transactivating capacity.

Fig. 11. Expression of p21 in response to TADp53. TADp53- and ptsp53-expressing
cells were incubated at the permissive (i.e., 31.5°C)
and the nonpermissive (i.e., 37°C) temperature. After
24 h, cells were subjected to Western blot using the mouse
monoclonal anti-p21 antibody WAF-1 Ab-1, as described in "Materials
and Methods." Arrow on the right,
position of p21 protein. Left, positions of molecular
weight standards (in thousands).

The apoptosis-inducing capacity of p53 does not necessarilydepend on
the transcriptional activity of p53 (37, 38, 39)
.To determine
whether transcriptionally deficient p53 can induceapoptosis in U-937
cells, ptsp53 and TADp53-expressing U-937clones and mock-transfected
control clones were incubated inculture medium at the optimal
temperature for the apoptosis-inducingactivity of ptsp53
(i.e., 31.5°C). Each day, cells werecounted, and
viability was assessed by trypan blue exclusion(Fig. 12)
. As shown, expression of ptsp53 conferred a rapidcell death to U-937
cells. In contrast, both mock-transfectedand TADp53 expressing
U-937 clones were viable throughout theexperiment, although
U-937/TADp53 clones were viable to a slightlylesser extent.
These data indicate that the main mechanism forp53-mediated apoptosis
in U-937 cells depends on the transcriptionregulatory capacity of p53.

Transcriptionally Deficient p53 Does Not Induce Differentiation of
U-937 Cells.
To determine whether p53-mediated differentiation of U-937 cells
isseparable from the transcriptional activity of p53,
TADp53-expressing,ptsp53 expressing, and mock-transfected U-937
clones were incubatedat the optimal temperature for the
differentiation-inducingactivity of ptsp53 (i.e., 32.5°C)
with or without 1 nM ofVit D3. As expected,
mock-transfected control U-937 clones showedno NBT reducing activity
when incubated in culture medium alone,whereas the U-937/ptsp53/A2
clone showed an NBT reducing activityof 14 ± 3%, reflecting the
differentiation inducing capacityof wild-type p53 per se
(Fig. 13)
and consistent with previousresults (13)
. However,
expression of TADp53 induced almostno signs of NBT reduction (<2%)
of the U-937 cells. The discrepancybetween U-937 clones expressing
wild-type p53 and transcriptionallydeficient p53 was even more
striking when incubated with VitD3. Although the U-937/ptsp53/A2 clone
had a pronounced differentiationresponse as measured by reduction of
NBT, the differentiationresponse of U-937/TADp53 clones was comparable
with the mock-transfectedcontrol clones, showing a low degree of NBT
reduction. Whenincubated at 31.5°C, the differentiation response of
U-937/TADp53clones was still comparable with the mock-transfected
controlclones (data not shown).

Fig. 13. Effects of TADp53 on induced differentiation, assayed by the capacity
to reduce NBT. Mock transfected (- Control) and
ptsp53expressing (+ Control) U-937 cells as well as
TADp53-expressing U-937 cells (TADp53) at an initial
concentration of 0.2 x 106 cells/ml were incubated at
32.5°C (i.e., the permissive, differentiation-inducing
ptsp53 temperature) with or without 1 nM Vit D3. After 4
days of incubation, the cells were subjected to NBT test. , culture
medium; , Vit D3 1 nM. Mean values from three separate
experiments are shown; bars, SE.

To extend the analysis of the differentiation-inducing capacityof
transcriptionally inactive p53, TADp53 expressing U-937 clonesand
positive and negative control clones incubated at the differentiation
permissivetemperature (i.e., 32.5°C) with or without Vit
D3 were screenedfor expression of the granulocyte/monocyte-related
cell surfaceantigen CD11c (50)
by a FACS analysis. As
shown in Table 5
,the differentiation response of the U-937 clones expressing
transcriptionallydeficient p53 was comparable with mock-transfected
control clonesboth with and without Vit D3. In contrast, wild-type p53
expressingU-937 cells responded with up-regulation of CD11c, which was
evenmore pronounced when incubated with 1 nM Vit
D3.

U-937 cells expressing transcriptionally deficient p53 and positive and
negative control U-937 cells were incubated at 32.5°C with or without
1 nM Vit D3. After 3 days, cells were analyzed for the
monocyte-related cell surface antigen CD11c by a FACS analysis. The
percentage of gated cells in one representative experiment is shown.

Thus, as judged by reduction of NBT and expression of the
differentiation-relatedcell surface antigen CD11c, transcriptionally
deficient p53is incapable of inducing or facilitating differentiation
ofU-937 cells. This indicates that the transcription regulatory
potentialof p53 is essential for p53-mediated differentiation.

Discussion

The tumor suppressor protein p53 has the potential to counteract
allthree features characterizing acute leukemia: the differentiation
block,the growth advantage, and the inhibition of apoptosis. However,
themechanisms for p53induced differentiation are largely unknown.
Theprotooncogene Bcl-2 inhibits a number of
p53-dependent activities.We demonstrate that although Bcl-2
partially inhibits p53-mediatedcell death, it does not interfere
with p53-mediated differentiation,suggesting that p53-induced
apoptosis and differentiation relyon, at least partly, separable
molecular mechanisms. Resultsfrom others indicate separable pathways
for p53-mediated cellcycle arrest and apoptosis (3, 51, 52, 53)
. Hence, althoughboth p53-mediated differentiation
and cell cycle arrest canbe separated from p53-mediated apoptosis, the
question as towhether p53-mediated differentiation can be separated
from p53-mediatedcell cycle arrest still remains to be answered.

Bcl-2 does not inhibit the induced differentiation of U-937cells,
regardless of expression of p53, in concert with earlierwork
suggesting that Bcl-2 does not interfere with differentiationinduced
by other means (19, 28, 29, 54)
. Moreover, althoughBcl-2
has been shown to inhibit cell proliferation (24, 25,26)
, we
did not observe any effects of Bcl-2 on the proliferationrate of U-937
cells. It is possible that a selection againstthe
G1 arresting aspects of Bcl-2 took place during
the initialstages of establishment of the clones, rendering cell
clonesunresponsive to the cell cycle regulatory properties of
Bcl-2.

Although we show that Bcl-2 inhibits p53-mediated apoptosis,the
protection against p53-mediated apoptosis is not complete.This might
be explained by distinct pathways from p53 leadingto cell death, in
line with previous reports indicating multiplepathways for
p53-mediated apoptosis (38)
. Accordingly, it hasbeen
shown that p53 can induce apoptosis in the absence of bax
(55)
.Moreover, the high background levels of bax in U-937
cells (datanot shown) might neutralize some of the overexpressed Bcl-2
protein,by these means reducing the levels of active Bcl-2.

The transcriptional activity of p53 has for long been the main
explanatorymechanism for the cellular effects of p53. However,
recentlyseveral reports have demonstrated that p53 can mediate both
apoptosisand cell cycle arrest independently of p53-mediated
transcriptionalactivation (35, 36, 37, 38, 56)
, bringing up a
role for p53not only as a transcription factor but also in direct
protein-proteinsignaling. Consequently, a p53-mediated,
differentiation-inducingroute independent from its transactivating
properties mightwell exist. For this purpose, we overexpressed TADp53
(41)
,a transcriptionally inactive p53 mutant in
monoblastic U-937cells. Although the capacity of TADp53 to activate
and represstranscription of genes is reduced severely (38, 41, 57)
,recent studies have demonstrated a weak ability to activate
reporterconstructs (48, 49)
. Our observation that TADp53
did neithermediate differentiation nor apoptosis in U-937 cells
indicatesthat the transcription regulatory potential of p53 is
essentialboth for induction of differentiation and of apoptosis in
U-937cells. Furthermore, these data are consistent with our finding
thatBcl-2 does not interfere with either the p53-mediated
differentiationinduction or the transcriptional activity of p53 in
U-937 cells.However, because the TADp53 used in this study has been
showndefective for p53-mediated repression as well as activation
(57)
,we cannot exclude that p53-mediated differentiation
and apoptosisrely on p53-mediated transcriptional repression.

Interestingly, the characteristics of the ptsp53(Val135) provide
furtherevidence for distinct pathways in p53-mediated cell death
versusdifferentiation. When incubated at 31.5°C, the cell
death-inducingand cell cycle-arresting features of ptsp53 dominate,
causingan almost complete cell death after 4 days. However, when
incubatedat 32.5°C, cell death is not observed. Instead, the
differentiation-relatedproperties of ptsp53 appear. These functional
differences mayreflect changes in the levels of wild-type conformation
p53,in that high levels of active p53 are required for apoptosis
induction,whereas lower levels might suffice for induction of
differentiation.This is in concordance with previous data showing high
levelsof p53 to induce apoptosis whereas low levels induce
differentiationof monoblastic HL-60 cells (58)
.
Accordingly, our own datademonstrate a slight but reproducible
reduction in the levelsof p53-induced p21 when U-937/ptsp53(Val135)
cells are incubatedat 32.5°C as compared with 31.5°C. We also show
thatthe transactivating capacity of ptsp53 is higher at 31.5°Cas
compared with 32.5°C, as measured by luciferase reporterassays.
However, because the luminescence in control incubationsperformed at
31.5°C was slightly elevated as compared withthe luminescence in
control incubations performed at 32.5°Cthroughout the experiments,
unspecific temperature-related effects,possibly influencing the
luminescence, cannot be excluded. Ithas been shown that ptsp53(Val135)
is located predominantlyin the cytoplasm at 37°C (i.e.,
the mutant conformation)but is imported into the nucleus at 32°C
(i.e., the wild-typeconformation; Ref. 59
). It
may well be that slight temperatureshifts around 32°C modulate the
precise amounts of activenuclear p53 protein.

In conclusion, Bcl-2 does not interfere with the p53-facilitated
differentiation,although it does inhibit p53-mediated apoptosis,
indicatingseparate molecular mechanisms in p53-mediated apoptosis
versusdifferentiation. Furthermore, our results indicate
that p53-mediateddifferentiation relies on the transcriptional
activity of p53in U-937 cells.

Materials and Methods

Cells and Culture Conditions.
The human monoblastic cell line U-937-4 (60)
and the
subcloneU-937ptsp53/A2 (13)
, expressing a murine
temperature-sensitiveform of p53 [i.e., ptsp53(Val135)]
(Refs. 61 and 62
), wascultured in RPMI 1640 (Life
Technologies, Inc., Paisley, UnitedKingdom), supplemented with 10%
heat-inactivated FCS (Life Technologies)in a humidified
CO2 atmosphere at 37°C. For wild-type activity
ofp53, cells were incubated at 31.5°C32.5°C. Thenumber of cells
and viability, as judged by trypan blue exclusion,were determined by
counting in a Bürker chamber. Exponentiallygrowing cells were
used for all experiments.

Vector Constructs.
The pGL3/Waf1/Luc vector (51)
, carrying 2.3 kb of
the p21 promotersequence in control of the firefly luciferase gene,
was kindlyprovided by professor Moshe Oren (Weizmann Institute
of Science,Rehovot, Israel). The pGL3/SV40/Luc vector, having the
fireflyluciferase gene under the control of the SV40 promoter, was
fromPromega Corp. (Madison, WI). The cDNA for human bcl-2
was generouslyprovided by Dr. Klas Wiman (Karolinska Institute,
Stockholm)and was cloned into the eukaryotic expression vector pCEP4.
pCEP4provides a CMV promoter-driven expression of Bcl-2 and confers
resistanceto hygromycin B, allowing for selection of recombinant
cells.The eukaryotic expression vector pMSVCl/p53(25,26,Val135) (Ref.
56
),carrying the cDNA for a murine transcriptionally
inactive doublemutant form of ptsp53(Val135) (Ref. 41
),
driven by the longterminal repeat from Harvey murine sarcoma virus,
was generouslyprovided by Professor Arnold Levine (Rockefeller
Institute,NY). p53(25,26,Val135) (TADp53) shows temperature-sensitive
DNAbinding (41)
as well as the original ptsp53(Val135),
but theresidues at amino acid positions 25 and 26 (corresponding to
humanLeu-22 and Trp-23, which bind to the TATA-associated factors
TAFII70and TAFII31 of the
transcriptional machinery) have been mutated,abolishing the
transactivating effects (1, 56)
. PMSVCl confersresistance
to geneticin, allowing for selection of recombinantcells
(63)
. To obtain control clones, U-937 cells and
U-937/ptsp53/A2cells were transfected with pMSVCl and pCEP4,
respectively.

Reporter Assays.
For transient transfection, cells were resuspended in 37°Cculture
medium (RPMI 1640 + 10% FCS) to a concentration of 20x
106 cells/ml. The plasmid was introduced into the
cells byelectroporation using the Bio-Rad gene-pulser (Bio-Rad,
Melville,NY) with electrical settings of 280 V and 960 µF, after
whichcells were incubated at 32.5°C or 31.5°C. After 16 h,
theluminescence of transfected cells was determined using the
Dual-LuciferaseReporter Assay System (Promega Corp., Madison, WI),
accordingto the manufacturers instructions. Briefly, transfected
cellswere washed in PBS and lysed in Passive Lysis Buffer (Promega)
underconstant agitation for at least 20 min. Twenty µl of lysate
weremixed with 100 µl of LARII (Promega), after which the
luminescenceof the firefly luciferase was measured in a TD 20/20
luminometer(Turner Designs, Sunnyvale, CA).

Transfection Procedure.
The transfection for constitutive protein expression was performedas
described previously (64)
. Cells were resuspended in
37°Cculture medium (RPMI 1640 + 10% FCS) to a concentration
of 5x 106 cells/ml. The plasmid was
introduced into the cells byelectroporation using the Bio-Rad
gene-pulser (Bio-Rad) withelectrical settings of 270 V and 960 µF.
After 2 days,cells were seeded with Geneticin at 1.5 mg/ml (Boehringer
Mannheim,Mannheim, Germany) or hygromycin B at 1.4 mg/ml
(Calbiochem-NovabiochemCorp., La Jolla, CA) in 96-well plates to allow
for selectionof transfected clones. After 23 weeks, individual cell
cloneswere expanded to mass cultures and assayed for expression of
TADp53by biosynthetic labeling, IP, and fluorography and for
expressionof Bcl-2 by IP-Western blot.

Assessment of Cell Surface Antigens by Flow Cytometric Analysis.
Cells were washed in PBS and resuspended to 510 x
106cells/ml 50 µl of the cell suspension was
incubated with5 µl of the following moAbs in microtiter wells:
controlIgG1-FITC/IgG1-PE, cd 11c-PE (Becton Dickinson, San José,
CA),Annexin V-FITC (PharMingen, San Diego, CA), and propidium iodide
(Sigma),for 10 min at room temperature under constant agitation. The
cellswere then washed three times and fixed in 1% paraformaldehyde
beforeflow cytometric analyses (FACSscan; Becton Dickinson). Ten
thousandcells were collected for each antibody. Dead cells and debris
wereexcluded from analysis by gating prior to the calculation ofthe
percentage of positive cells, using the control incubationwith
IgG1-FITC/IgG1-PE for marker settings. Cells were analyzedfor
expression of Annexin V in parallel with staining with propidium
iodide,which makes it possible to exclude necrotic cells. This
providesa selective method for detection of apoptosis
(46)
.

We thank Tor Olofsson and the staff at the ImmunoCytoHematology
Laboratoryfor valuable discussions and for skillfully
performing the analysisof cell cycle distribution and cell surface
antigens by flowcytometry. We also thank Olga Göransson for help
with densitometricanalyses.

Footnotes

The costs of publication of this article were defrayed in partby the payment of page charges. This article must thereforebe hereby marked advertisement in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

1 This work was supported by the Swedish Cancer
Foundation, theSwedish Childhood Cancer Foundation, The Tobias
Foundation,The Swedish Medical Research Council (Project 11546), Funds
ofLunds sjukvårdsdistrikt, and the Gunnar, Arvid and Elisabeth
NilssonFoundation.